U.S. patent application number 10/015593 was filed with the patent office on 2002-07-11 for two-color differential display as a method for detecting regulated genes.
This patent application is currently assigned to Aventis Pharma Deutschland GmbH.. Invention is credited to Kozian, Detlef, Reuner, Birgit.
Application Number | 20020090636 10/015593 |
Document ID | / |
Family ID | 7880045 |
Filed Date | 2002-07-11 |
United States Patent
Application |
20020090636 |
Kind Code |
A1 |
Kozian, Detlef ; et
al. |
July 11, 2002 |
Two-color differential display as a method for detecting regulated
genes
Abstract
The invention relates to a novel method for analyzing the
composition of an mRNA sample and analyzing differential gene
expression using two differently labeled primers, and to the use of
the method. In the method for analyzing an RNA sample, a) a first
primer, which is, where appropriate, labeled with a first dye, is
used to prepare the first strand of a complementary DNA sample
(cDNA sample) from an RNA sample, b) a second primer, which is
preferably labeled with a second dye, is used to prepare the second
strand of this cDNA sample, c) the first primer, which is labeled
with a first dye, and the second primer, which is labeled with a
second dye, are used to amplify the cDNA sample, and d) the
composition of the amplified, labeled cDNA sample is analyzed.
Inventors: |
Kozian, Detlef; (Munchen,
DE) ; Reuner, Birgit; (Munchen, DE) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT &
DUNNER LLP
1300 I STREET, NW
WASHINGTON
DC
20005
US
|
Assignee: |
Aventis Pharma Deutschland
GmbH.
|
Family ID: |
7880045 |
Appl. No.: |
10/015593 |
Filed: |
December 17, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10015593 |
Dec 17, 2001 |
|
|
|
09390324 |
Sep 7, 1999 |
|
|
|
Current U.S.
Class: |
435/6.12 ;
435/91.2 |
Current CPC
Class: |
C12Q 1/6853 20130101;
C12Q 1/6809 20130101; C12Q 2565/102 20130101; C12Q 2525/173
20130101; C12Q 2525/173 20130101; C12Q 2521/107 20130101; C12Q
2565/102 20130101; C12Q 1/6853 20130101; C12Q 2521/107 20130101;
C12Q 1/6809 20130101; Y10T 436/143333 20150115 |
Class at
Publication: |
435/6 ;
435/91.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 7, 1998 |
DE |
19840731.9 |
Claims
1. A method for analyzing an RNA sample, which comprises a) using a
first primer, which is, where appropriate, labeled with a first
dye, to prepare the first strand of a complementary DNA sample
(cDNA sample) from an RNA sample, b) using a second primer, which
is preferably labeled with a second dye, to prepare the second
strand of this cDNA sample, c) using the first primer, which is
labeled with a first dye, and the second primer, which is labeled
with a second dye, to amplify the cDNA sample, and d) analyzing the
composition of the amplified, labeled cDNA sample.
2. The method as claimed in claim 1, wherein the first primer
contains an oligo (dT) sequence.
3. The method as claimed in one or both of claims 1 and 2, wherein
the oligo (dT) sequence of the first primer is composed of at least
10 thymidine nucleotides.
4. The method as claimed in one or more of claims 1 to 3, wherein
the first primer contains, at the 3' end of the oligo (dT)
sequence, at least two further nucleotides, which do not belong to
the oligo (dT) sequence.
5. The method as claimed in one or more of claims 1 to 4, wherein
the first primer is a heterogeneous mixture of primer molecules
which differ in the sequence of the nucleotides which are in
positions M or N and which do not belong to the oligo (dT)
sequence.
6. The method as claimed in one or more of claims 1 to 5, wherein
the second primer is at least 6 nucleotides in length.
7. The method as claimed in one or more of claims 1 to 6, wherein
the first and second dyes are different fluorescences.
8. The method as claimed in one or more of claims 1 to 7, wherein
the fluorescences are selected from the fluorescences Cy2, Cy3,
Cy5, FAM, 6-FAM, FITC, fluorescein, HEX, 5-IAF, TAMRA, TET, XRITC,
ROX, Alexa488, Alexa532, Alexa546, Alexa594, Texas red and
lissamine.
9. The method as claimed in one or more of claims 1 to 8, wherein,
apart from a first RNA sample, one or more additional RNA samples
are analogously compared analytically.
10. The use of a method as claimed in one or more of claims 1 to 9
for analyzing differential gene expression.
11. The use of a method as claimed in one or more of claims 1 to 9
for identifying and/or characterizing pharmacological active
compounds.
12. The use of a method as claimed in one or more of claims 1 to 9
for identifying target genes.
Description
[0001] The invention relates to a novel method for analyzing the
composition of an mRNA sample and analyzing differential gene
expression using two differently labeled primers, and to the use of
the method.
[0002] Differential RNA display (DD) is one of the methods which is
most frequently used for detecting and isolating regulated genes
(Liang, P. and Pardee, A. B. (1992) Science 257, 967-971; Liang and
Pardee (1995) Current Opinion in Immunology 7, 274-280; McClelland
et al. (1995) Trends in Genetics 11: 242-246). The DDRT
(differential display+reverse transcription: DDRT) method involves,
in a first step, reverse transcribing isolated RNA using a first
reverse primer (reverse transcription (RT)), thereby preparing the
first strand of a complementary DNA (cDNA), then amplifying this
DNA, in a second step, by the polymerase chain reaction (PCR) using
the first primer and a second primer, and then analyzing the
composition of the amplified cDNA sample, for example by
fractionating the amplified cDNA in a gel. While detection can be
effected, for example, by hybridizing with a labeled probe or by
labeling the amplified cDNA, it can also be effected by carrying
out the amplification in the presence of radioactively labeled
nucleotides (usually radioactively labeled dATP) or in the presence
of a labeled primer which is, for example, labeled with a
fluorescence ("fluorescence DDRT") (Ito et al. (1994) FEBS Letters
351, 231-236; Ito and Sakaki ("Methods in Molecular Biology Vol.
85: Differential Display Methods and Protocols" (1997) p. 37-44
Liang and Pardee eds, Human Press Inc. Totowa, N.J.; Jones et al.
(1997) Biotechniques 22, 536-543; Smith et al. (1997) Biotechniques
23, 274-279).
[0003] If the reaction is carried out in the presence of
radioactively labeled nucleotides, all the amplified cDNAs are then
labeled (radioactive DDRT; classical DDRT). If, on the other hand,
a labeled primer is used (e.g. fluorescence DDRT), only a part of
the amplified cDNAs is labeled, as a result of the possible primer
combinations of the first labeled primer and of the unlabeled
second primer during the PCR, while another part remains
unlabeled.
[0004] The first labeled primer (in this case, primer 1 is an
oligo(dT) primer which possesses two further nucleotides (M=A, C,
G; N=A, C, G, T) at the 3' end ("5'-(T).sub.12-MN-3'" is
(T).sub.12-MN)) is used for the RT. In the subsequent PCR, a
second, unlabeled primer (in this case, primer 2 is an
oligonucleotied which has a sequence of 10 randomly ordered
nucleotides ("10mer")) is used in addition to this first primer;
however, the first primer is customarily used in a twofold excess.
In this way, a relatively high proportion of amplified cDNAs whose
3' ends exhibit the sequence of the first primer and whose 5' ends
exhibit the sequence of the second primer (e.g. 5'-10mer ------
(T).sub.12-MN-3', where "------ " symbolizes the amplified cDNA
sequence which is located between the primer sequences) is obtained
as a rule. In addition to this, cDNAs are amplified in which the
primer sequences are transposed as compared with 1) (at whose 5'
and 3' ends can be found the sequences of the (T).sub.12-MN primer
and of the 10mer primer, respectively; cf. 2)). Other cDNAs are
either amplified only using the (T).sub.12-MN primer (cf. 3) or
only using the 10mer primer (cf. 4). Consequently, when two
different primers are used, it is possible to amplify the following
reaction products in a DDRT as a result of the possible primer
combinations:
1 1) 5'-10mer (T).sub.12-MN-3' 2) 5'-(T).sub.12-MN 10mer-3' 3)
5'-(T).sub.12-MN (T).sub.12-MN-3' 4) 4) 5'-10mer 10mer-3'
[0005] Owing to the fact that only the first primer is labeled in
this fluorescence DDRT, only those cDNAs which were amplified in at
least one direction using the labeled first primer, i.e. the cDNAs
specified under 1), 2) and 3), are detected in the subsequent
analysis of the amplified cDNAs, whereas cDNAs which are only
amplified using the second primer are neither labeled nor detected
(cf. 4). Consequently, the complexity of the detected PCR products
(cDNAs) is less than that in a radioactive DDRT. For this reason,
the conventional fluorescence DDRT does not detect those regulated
genes, or the mRNAs which correspond to them, which are only
amplified using the second primer. Consequently, the number of
regulated genes which is detected by the conventional fluorescence
DDRT can be lower than that detected in a radioactive DDRT.
[0006] Furthermore, when one single mRNA is used, a uniform cDNA
product is not amplified in the PCR either with conventional
radioactive DDRT or with conventional fluorescence DDRT, as shown
under 1) to 4); instead, several cDNAs, which differ in their
length (this would be evident, for example, by fractionating in a
gel), can be amplified as a result of the different primer
combinations. Only a detailed analysis (e.g. a sequence analysis)
would show whether different amplified cDNAs are cDNA products of
one and the same regulated gene or not. The conventional DDRTs do
not provide the possibility of differentiating between the
redundant labeled, amplified cDNAs. In a radioactive DDRT, it is
not possible to differentiate between 1) to 4). In a conventional
fluorescence DDRT, it is not possible to differentiate between 1),
2) and 3), although there is a greater probability that the visible
cDNA fragment derives from the 3' region of a gene since the 3'
primer is labeled with the fluorescence dye.
[0007] Consequently, the redundancy, on the one hand, of amplified
cDNA fragments in conventional radioactive DDRT, which redundancy
cannot be established or can only be established with a relatively
large amount of effort, and, on the other hand, the lower
complexity of the amplified cDNA product in conventional
fluorescence DDRT as compared with conventional radioactive DDRT,
with the risk of overlooking particular regulated genes, are the
main problems of conventional radioactive DDRT and conventional
fluorescence DDRT methods, respectively.
[0008] The invention on which the present application is based
provides a method which can be used for analyzing RNA samples and
in particular for analyzing differentially regulated genes, which
method does not suffer from the abovementioned disadvantages.
[0009] The present invention relates to a method for analyzing an
RNA sample, preferably an mRNA sample, which comprises
[0010] a) using a first primer, which is, where appropriate,
labeled with a first dye, to prepare the first strand of a
complementary DNA sample (cDNA sample) from an RNA sample,
preferably an mRNA sample,
[0011] b) using a second primer, which is preferably labeled with a
second dye, to prepare the second strand of this cDNA sample,
[0012] c) using the first primer, which is labeled with a first
dye, and the second primer, which is labeled with a second dye, to
amplify the cDNA sample, and
[0013] d) analyzing the composition of the amplified and labeled
cDNA sample.
[0014] The RNA sample contains mRNA to be analyzed. In a preferred
embodiment of the invention, mRNA is used in the method. The RNA
can be isolated, for example, by known methods such as CsCl.sub.2
density gradient centrifugation or column chromatography. The RNA
is preferably isolated from a cell or a population of cells or from
tissue. Where appropriate, the mRNA can be enriched from the RNA,
for example by means of chromatrography through an oligo (dT)
column.
[0015] The method employs a first primer and a second primer. The
first primer is the primer which is used for the reverse
transcription. The first primer can also be designated the 3'
primer since it hybridizes with the mRNA and defines the 3' end of
the first strand of the complementary DNA (cDNA). The second primer
is used to synthesize the second strand of the cDNA. The second
primer is also designated the 5' primer since it hybridizes with
the first strand of the cDNA and defines the 5' end of the second
strand of the cDNA.
[0016] Where appropriate, the first primer and the second primer
are labeled with a dye. The first primer is used for the reverse
transcription (procedural step a)) and for the subsequent
amplification of the cDNA (procedural step c)). The first primer,
which is used for the RT, is either labeled with a dye or not
labeled with a dye; the latter is, for example, the case when the
reverse transcriptase employed does not accept the labeled primer.
The first primer is in any case labeled when it is used for
amplifying the cDNA. The first primer which is used for the RT and
the first primer which is used for the amplification preferably
have the same sequence.
[0017] The second primer, which is used both for synthesizing the
second strand of the cDNA (procedural step b)) and for amplifying
the cDNA (procedural step c)), is preferably labeled both during
the second strand synthesis and during the amplification. The
second primer preferably has the same sequence on both occasions.
The second strand synthesis and the amplification of the cDNA are
preferably elements of one reaction such that the second primer is
identical.
[0018] In preferred embodiments of the invention, the first and
second primers are oligodeoxynucleotides which are composed of
deoxyadenosine (dA), deoxyguanosine (dG), deoxyinosine (dI),
deoxyuridine (dU), (deoxy)thymidine (dT) and deoxycytidine
(dC).
[0019] The first and/or the second primer can have a base sequence
(sequence composed of adenine, guanine, cytosine and thymine;
termed "sequence" in that which follows) which is complementary to
the sequence(s) of one (or more) particular nucleic acid(s). A
primer which is complementary to a particular sequence can
hybridize with a nucleic acid which contains this complementary
sequence or a sequence which is derived from this complementary
sequence (that is exhibits a certain homology with this
complementary sequence). For example, the first primer can
preferably hybridize with the mRNA. The first strand of the cDNA is
generated by primer extension. A primer can also have a sequence
which is identical, or essentially identical, to the sequence of a
particular nucleic acid. This primer can then hybridize (under
specific reaction conditions) with a nucleic acid which has a
sequence which is complementary to that of the particular nucleic
acid. For example, the sequence of the second primer is derived
from the sequence of the mRNA, i.e. the sequence of the second
primer can be identical or essentially identical to the mRNA
sequence. The second primer can therefore (under specific reaction
conditions) hybridize with a nucleic acid which possesses a
sequence which is complementary to the sequence of the mRNA, e.g.
the first strand of the cDNA.
[0020] The reaction conditions under which the first primer and the
second primer hybridize with a nucleic acid are preferably specific
temperature and/or buffer conditions. The temperature conditions
are preferably chosen such that the primers hybridize specifically
with the complementary nucleic acid sequence, i.e. such that it is
predominantly "correct" base pairings (A-T; G-C) which take place,
with as few incorrect base pairings as possible, i.e., if possible,
an "optimal" hybridization temperature (annealing temperature) is
chosen (e.g. Sambrook et al. (1989) Cold Spring Harbor Laboratory
Press). In addition to this, specific salt concentrations, in
particular Mg.sup.2+ concentrations, are chosen.
[0021] A nucleic acid from whose sequence the sequence(s) of the
first and/or second primer is/are derived (i.e. to which this/these
sequence(s) is/are complementary or identical or essentially
identical) can, for example, be a DNA, e.g. a cDNA, a gene, or a
part thereof, or an RNA, preferably an mRNA.
[0022] The second primer can possess a random base sequence, with
this encompassing both primers which possess a completely random
sequence and primers which possess a partially random sequence
(e.g. random in relation to one or more bases). While the sequences
may be random, they may at the same time exhibit a particular ratio
between the individual bases, e.g. all the bases may occur in the
same ratio or one or more bases may be over-represented or
under-represented. In particular, hypoxanthine can also be
used.
[0023] The second primer can also possess one or more defined base
sequences. This/these defined sequence(s) may be selected randomly,
i.e. they do not derive from known sequences. Primers of this
nature can be used to identify new genes or proteins which have not
previously been identified.
[0024] The second primer can possess a base sequence which is
derived from known sequences, i.e. which corresponds entirely or
partially to known sequences (i.e. which is complementary or
identical or essentially identical to these sequences). For
example, a primer may possess a sequence which exhibits a greater
or lesser degree of congruence with a particular consensus sequence
or else corresponds to this sequence. This approach can be used,
for example, to identify differential expression of known and/or
unknown members of a particular gene family/protein family.
[0025] The second primer can possess a base sequence which is
derived from a particular amino acid sequence (e.g. a consensus
sequence at the amino acid level). In this case, the primers can be
degenerate, i.e. allowance is made for the degeneracy of the
genetic code in relation to a particular amino acid sequence; the
primer then constitutes a mixture of primer molecules which possess
different sequences and allow for all the codon usage possibilities
for encoding the corresponding amino acid sequence (degenerate
primer).
[0026] The sequence of the second primer preferably possesses a
restriction cleavage site for a restriction endonuclease.
[0027] The second primer can preferably have a length of from 8 to
20 nucleotides in order to ensure that hybridization is as specific
as possible. However, the length is preferably selected in
accordance with the particular primer sequences and/or the reaction
conditions. The lengths and sequences of the first and second
primer are chosen independently of each other.
[0028] The first and/or second primer(s) can also contain (a)
sequence(s) which is/are not required for the hybridization and/or
which does/do not contribute to the hybridization. Other sequences
of this nature can, for example, make possible or facilitate the
further characterization or use of the amplified cDNA. These other
sequences are preferably located at the 5' end of the primer. For
example, the primer may contain a sequence which facilitates
subsequent sequencing e.g. an M13 sequence, and/or a sequence which
facilitates the preparation of a labeled probe (e.g. for
hybridizing Northern blots or Southern blots and/or for hybridizing
in situ), e.g. a T7 promoter sequence, a T3 promoter sequence or an
SP6 promoter sequence.
[0029] In the first procedural step, the mRNA is
reverse-transcribed, thereby preparing the first strand of a cDNA
(procedural step a)). A first primer, which is labeled with a dye,
where appropriate, is used for the RT. The first primer preferably
possesses an oligo(dT) sequence ("(T).sub.X", where "X" indicates
the number of thymidine residues) which hybridizes with the poly
(A) tail of each of the mRNAs and is in this way able to provide a
free 3'-OH end for the polymerase reaction. The oligo (dT) sequence
is preferably a sequence composed of 10-20 thymidine residues
(X=10-20), with a sequence composed of 12 thymidine residues (X=12)
or 15 thymidine residues (X=15) being particularly preferred.
[0030] In a particular preferred embodiment, the sequence of the
first primer possesses, at the 3' end of the oligo (dT) sequence,
at least one further nucleotide, preferably, however, two
nucleotides, which does/do not belong to the oligo (dT) sequence,
i.e. denoting that the first nucleotide which is joined on to the
3' end of the oligo (dT) sequence is different from thymidine. The
first primer preferably constitutes a mixture of primer molecules
which differ in the sequence of the nucleotides which do not belong
to the oligo (dT) sequence. For example, the first primer can have
the sequence 5'-(T).sub.XMN-3', where "M" is A (the base adenine),
C (the base cytosine) or G (the base guanine) and "N" is A, C, G or
T (the base thymine). For example, X can=12 or 15:
[0031] SEQ ID NO. 1: 5'-TTTTTTTTTTTTMN-3'
(5'-(T).sub.12-MN-3'"),
[0032] SEQ ID NO. 2: 5'-TTTTTTTTTTTTTTTMN-3'
(5'-(T).sub.15-MN-3'"),
[0033] where
[0034] "M" is A, C or G, and
[0035] "N" is A, C, G or T.
[0036] The first primer preferably constitutes a mixture of
different primers which differ from each other in the sequence
which does not belong to the oligo (dT) sequence.
[0037] 5'-T.sub.(X)MN-3' can be a mixture of primer molecules
having the sequences:
[0038] 5'-T.sub.(X)AN-3'
[0039] 5'-T.sub.(X)CN-3'
[0040] 5'-T.sub.(X)GN-3'
[0041] in which N=A, C, G, T
[0042] 5'-T.sub.(X)MN-3' is preferably a mixture of primer
molecules having the sequences:
[0043] 5'-T.sub.(X)MA-3'
[0044] 5'-T.sub.(X)MC-3'
[0045] 5'-T.sub.(X)MG-3'
[0046] 5'-T.sub.(X)MT-3'
[0047] in which M=A, C, G,
[0048] where the possible bases A, C and G are represented
uniformly in the mixture; for example, 5'-(T).sub.XMN-3' is a
mixture of primer molecules having the following sequences:
5'-(T).sub.XAG, 5'-(T).sub.X CG, 5'-(T).sub.X GC, 5'-(T).sub.X AT,
5'-(T).sub.X CT, 5'-(T).sub.X GT, etc. It is possible in this way
to provide primers for amplifying different mRNAs of unknown
sequence. At the same time, this arrangement ensures that the
primers preferentially hybridize with the 3' end of the
complementary sequence (at the poly (A) tail of the mRNA).
[0049] An RNA-dependent DNA polymerase, for example the enzyme
reverse transcriptase or another DNA-dependent polymerase having
reverse transcriptase activity, e.g. an appropriate
temperature-stable polymerase which can then also be used for
subsequently amplifying the cDNA (procedural step c)) is preferably
used for the RT. In a particular embodiment of the invention, the
synthesis of the second strand of the cDNA is an element of the
procedural step for amplifying the cDNA, with the procedural step
for amplifying the cDNA preferably being a PCR reaction.
[0050] The reverse transcription can, for example, be carried out
at 37.degree. C.-50.degree. C., preferably at 40.degree.
C.-45.degree. C., particularly preferably at 42.degree. C.
Preference is given to using a hybridization temperature which
enables the first primer to hybridize specifically (as few
incorrect base pairings as possible), which ensures that the
activity of the polymerase is sufficiently high and which makes it
possible to obtain transcripts which are as complete as
possible.
[0051] The second strand of the cDNA is synthesized using a second
primer which is preferably labeled. The second primer is preferably
at least 6 nucleotides in length (a 6mer), particularly preferably
from 10 (a 10mer) to 20 (a 20mer) nucleotides in length. In a
particular embodiment of the invention, the second primer is 13
nucleotides in length (a "13mer"). For example, the sequence of the
second primer can be "(N).sub.X", where "X" is 6-20 and where "N"
is A, C, G or T, independently of each other.
[0052] The first and second primers are preferably synthetic
oligonucleotides which can, for example, be obtained commercially
or can be synthesized on a solid phase using a known method (e.g.
the phosphoramidite method in accordance with Caruthers et al.
(1983) Tetrahedron Letters 24, 245). The primers are preferably
composed of the nucleotides deoxyadenosine, deoxyguanosine,
deoxyinosine, deoxycytidine and deoxythymidine.
[0053] In a particularly preferred embodiment of the method, the
synthesis of the second strand of the cDNAs is already an element
of the PCR reaction, with this strand being synthesized under the
reaction conditions under which the PCR is carried out.
[0054] The first and second primers are preferably labeled
differently. In principle, any type of labelling can be used, for
example a primer can be coupled to digoxigenin or biotin such that
a cDNA molecule which has been amplified using this primer can be
detected, e.g. with an appropriate antibody or enzyme, or a primer
can be coupled to a chemical compound which enables a substrate to
be converted after it has been added, e.g. Atto-Phos system (JBL
Scientific, San Luis Obispo, Calif., USA) or ECF substrate
(Amersham-Pharmacia Biotech, Freiburg, Germany).
[0055] However, particular preference is given to labelling the
first and/or second primer with fluorescences. Preference is given
to labelling the first primer with a first fluorescence and the
second primer with a second fluorescence. The first and second
fluorescences are preferably chosen such that the two fluorescences
employed can be clearly differentiated. In this context, the
unambiguity of the result, i.e. the detectable difference between
the labelling patterns of individual cDNAs, depends both on the
fluorescences employed and on the sensitivity of the analytical
method. As a rule, computer-assisted analytical methods are used
for evaluating the results. For example, the fluorescences can be
selected such that the detectable excitation wavelengths and/or
emission wavelengths of the first and second fluorescences differ
by 200 nm or more, e.g. 250 nm, 300 nm, 350 nm or 400 nm.
[0056] Examples of fluorescences to which the first and/or second
primer can be coupled are Cy2, Cy3, Cy5, FAM, 6-FAM
(6-carboxyfluorescein (blue)), FITC (fluorescein isothiocyanate),
fluorescein, HEX (4, 5, 2',4',5',7'-hexachloro-6-carboxyfluorescein
(green)), 5-IAF, TAMRA (6-carboxytetramethylrhodamine (yellow)),
TET (4, 7, 2', 7'-tetrachloro6-carboxyfluorescein), XRITC
(rhodamine-X-isothiocyanate), ROX (6-carboxyrhodamine (red)),
Alexa488, Alexa532, Alexa546, Alexa594, Texas red and
lissamine.
[0057] For example, the following combinations are possible:
[0058] Primer 1 labeled with Cy5 and primer 2 labeled with
Alexa488; primer 1 labeled with Cy5 and primer, 2 labeled with
FITC; primer 1 labeled with Cy5 and primer 2 labeled with FAM;
primer 1 labeled with Cy5 and primer 2 labeled with Cy2; primer 1
labeled with Cy5 and primer 2 labeled with Cy3; primer 1 labeled
with fluorescein and primer 2 labeled with Texas red; primer 1
labeled with fluorescein and primer 2 labeled with lissamine;
primer 1 labeled with fluorescein and primer 2 labeled with ROX;
primer 1 labeled with fluorescein-Cy3; primer 1 labeled with
Alexa594 and primer 2 labeled with Alexa488; primer 1 labeled with
Alexa568 and primer 2 labeled with Alexa488; primer 1 labeled with
Alexa546 and primer 2 labeled with Alexa488; primer 1 labeled with
Alexa532 and primer 2 labeled with Alexa488; primer 1 labeled with
Texas red and primer 2 labeled with Alexa488; primer 1 labeled with
ROX and primer 2 labeled with Alexa488; primer 1 labeled with
Alexa488 and primer 2 labeled with lissamine; primer 1 labeled with
Alexa488 and primer 2 labeled with Cy3; primer 1 labeled with
Alexa488 and primer 2 labeled with ROX. Naturally, corresponding
primer combinations, in which the fluorescences used for the
labelling are transposed, with regard to primer 1 and primer 2, as
compared with the above examples, are also possible.
[0059] The fluorescences are preferably coupled to the 5' end of
the primer (oligonucleotide). In the case of an oligo (dT) primer,
a further nucleotide which is different from thymidine, for example
a guanosine, can be located at the 5' end in front of the
fluorescence. In addition, a fluorescence can also be coupled to
the oligonucleotide by way of a base. Where appropriate, a
fluorescence can also be coupled to the first and/or second primer
by way of a suitable linker.
[0060] Examples of labeled primers which can be used as the first
and/or second primer are:
[0061] 5'-CY5-G(T).sub.15MN-3', 5'-CY2-G(T).sub.15MN-3',
5'-CY3-G(T).sub.15MN-3',
[0062] 5'-FAM-G(T).sub.15MN-3', 5'-6-FAM-G(T).sub.15MN-3',
5'-FITC-G(T).sub.15MN-3',
[0063] 5'-fluorescein-G(T).sub.15MN-3', 5'-HEX-G(T).sub.15MN-3',
5'-5-IAF-G(T).sub.15MN-3',
[0064] 5'-TAMRA-G(T).sub.15MN-3', 5'-TET-G(T).sub.15MN-3',
5'-XRITC-G(T).sub.15MN-3',
[0065] 5'-ROX-G(T).sub.15MN-3', 5'-Alexa488-G(T).sub.15MN-3',
5'-Alexa532-G(T).sub.15MN-3',
[0066] 5'-Alexa546-G(T).sub.15MN-3',
5'-Alexa594-G(T).sub.15MN-3',
[0067] 5'-Texas red-G(T).sub.15MN-3', 5'-lissamine-G(T).sub.15MN-3'
(the primers which have so far been mentioned are preferably used
as first primers), 5'-CY5-(N).sub.13-3',
[0068] 5'-CY2-(N).sub.13-3', 5'-CY3-(N).sub.13-3',
5'-FAM-(N).sub.13-3', 5'-6-FAM-(N).sub.13-3',
[0069] 5'-FITC-(N).sub.13-3', 5'-fluorescein-(N).sub.13-3',
5'-HEX-(N).sub.13-3',
[0070] 5'-5-IAF-(N).sub.13-3', 5'-TAMRA-(N).sub.13-3',
5'-TET-(N).sub.13-3',
[0071] 5'-XRITC-(N).sub.13-3', 5'-ROX-(N).sub.13-3',
5'-Alexa488-(N).sub.13-3',
[0072] 5'-Alexa532-(N).sub.13-3', 5'-Alexa546-(N).sub.13-3',
5'-Alexa594-(N).sub.13-3',
[0073] 5'-Texas red-(N).sub.13-3'and
5'-lissamine-(N).sub.13-3'(these primers are preferably used as
second primers),
[0074] where
[0075] "M" is A, C or G and
[0076] "N" is A, C, G or T.
[0077] When the first and/or second primer is/are labeled with a
fluorescence, care must be taken to ensure that the excitation
wavelengths are sufficiently far apart in order to be able to
establish, after the excitation, to which wavelength an observable
emission was due. An example of a good combination is a Cy5
labelling (excitation at 650 nm), e.g. of the first primer (e.g. of
a TX-MN primer), and a fluorescein-based labelling (exitation at
490 nm) of the second primer. The use of two differently labeled
primers makes it possible to draw an unambiguous conclusion about
the primer composition of (a) detected amplified cDNA(s), e.g. in a
gel.
[0078] If, for example, only an emission at 650 nm can be observed,
the cDNA was then amplified using the Cy5-labeled primer and this
cDNA has the first primer (e.g. the (T).sub.X-MN primer) at one end
at least. If an emission can be observed at an excitation
wavelength of 490 nm, at least one end of the cDNA is provided with
the second primer. If a particular amplified cDNA (in the gel) only
emits at one excitation wavelength, then the cDNA was only
amplified, in the amplification, using one primer, specifically the
primer having a fluorescence which emits at the corresponding
excitation wavelength. If a particular amplified cDNA (in the gel)
emits at two excitation wavelengths, this cDNA was then amplified
using the first and/or the second primer.
[0079] The amplification (preferably PCR) is carried out using a
DNA polymerase, preferably a DNA-dependent DNA polymerase, with
particular preference being given to this DNA polymerase being a
temperature-stable DNA polymerase, e.g. Taq polymerase, VENT
polymerase, AmpliTaq polymerase or AmpliTaq Gold polymerase, inter
alia. The PCR is carried out using a temperature profile which
preferably enables the cDNA to be amplified exponentially. For
example, the temperature cycle quoted in Example 1 can be used for
this purpose. Preference is given to running 30-40 or more
cycles.
[0080] After that, the labeled and amplified cDNA is analyzed. For
example, the amplified cDNA can be fractionated in a gel, with
amplified cDNAs which differ in their length being located in
different bands. The further analysis of these bands can take
place, for example, in scanners which excite the fluorescence at a
given wavelength (which depends on the fluorescence employed in
each case) and/or which are able to detect the light which is
emitted in association with the chemical transformation of a
chemiluminescent substrate. The emitted fluorescent light can then,
for example, be evaluated with an appropriate computer program and
assembled into a gel picture (as is known, for example, from
autoradiographs). Examples of scanners which can be used are the
Fluorlmager 575, Fluorimager SI, Fluorlmager (Molecular Dynamics,
Krefeld, Germany), Strom (Molecular Dynamics), FLA-2000 (Fuji,
Tokyo, Japan), FMBioll (Hitachi, through Biozym Diagnostic GmbH,
Hess, Oldendorf, Germany), Fluor-S-Multilmager and Molecular Imager
FX (BioRad, Munich, Germany) scanners.
[0081] The method employs, in particular, mRNA (also termed "mRNA
sample") which is isolated from a cell. As a rule, a sample of this
nature is a heterogeneous mRNA sample (i.e. it contains a mixture
of the mRNAs which were present in this cell at the time the RNA
was isolated). As a rule, this heterogeneous mRNA sample represents
the genes which were expressed by a particular cell at a particular
time and under particular conditions, i.e. the composition of the
mRNA sample depends, for example, on the cell type, its state of
differentiation, the cell cycle and/or the previous treatment of
the cell, etc.
[0082] One particular embodiment of the invention relates to the
analysis of a heterogeneous mRNA sample, which comprises
[0083] a) using a first primer, which is, where appropriate,
labeled with a first dye, to prepare the first strand of a
complementary heterogeneous DNA sample (heterogeneous cDNA sample)
from a heterogeneous mRNA sample,
[0084] b) using a second primer, which is preferably labeled with a
second dye, to prepare the second strand of this heterogeneous cDNA
sample,
[0085] c) using the first primer, which is labeled with a first dye
("labeled first primer"), and the second primer, which is labeled
with a second dye ("labeled second primer"), to amplify the
heterogeneous cDNA sample, and
[0086] d) analyzing the composition of the heterogeneous, amplified
and labeled cDNA sample.
[0087] The genes which are expressed by a particular cell at a
particular time which are especially of interest are those which
are differentially expressed or regulated. In the present case, it
is in particular the analytical comparison of differentially
expressed genes which is of interest. One particular embodiment of
the invention relates to a correspondingly analogous method for
analytically comparing two or more heterogeneous mRNA samples.
[0088] One particular embodiment of the invention relates to a
method for analytically comparing a first heterogeneous mRNA sample
with one or more additional heterogeneous mRNA samples, which
comprises
[0089] a) using a first primer, which is, where appropriate,
labeled with a first dye, for each of two or more heterogeneous
mRNA samples, to prepare, in each case, the first strand of a
complementary, heterogeneous DNA sample (heterogeneous cDNA sample)
from these mRNA samples,
[0090] b) then using a second primer, which is preferably labeled
with a second dye, to prepare, in each case, the second strand of
the heterogeneous cDNA sample for each of these samples,
[0091] c) using the first labeled primer and the second labeled
primer to amplify each of the heterogeneous cDNA samples, and
[0092] d) analyzing the composition of each heterogeneous,
amplified and labeled cDNA sample and comparing the compositions of
the samples.
[0093] Another particular embodiment of the invention relates to a
method which comprises
[0094] a) using a first primer, which is, where appropriate,
labeled with a first dye, for each of two or more heterogeneous
mRNA samples, to prepare, in each case, the first strand of a
complementary, heterogeneous DNA sample (heterogeneous cDNA sample)
from these mRNA samples,
[0095] b) using a second primer, which is preferably labeled with a
second dye, to prepare, in each case, the second strand of the
heterogeneous cDNA sample for each of the samples,
[0096] c) using the first labeled primer and the second labeled
primer to amplify each of the heterogeneous cDNA samples, and
[0097] d) carrying out an analysis to determine which of the
samples does/do contain or not contain individual mRNA
molecules.
[0098] After the cDNA has been amplified and the composition of the
cDNA has, where appropriate, been analyzed, an investigation is
carried out to determine which of the amplified cDNAs have which
primer composition, i.e. which cDNAs were amplified using only the
first primer, which were amplified using only the second primer and
which were amplified using the first and the second primers
(affiliation of the differentially amplified cDNAs to groups 1)+2),
3) and 4)). The invention also relates to follow-up methods in
which particular amplified cDNAs are selected on the basis of the
primer composition of the amplified cDNAs and, where appropriate,
subjected to further analysis and/or further use. In addition to
this, the invention relates to follow-up methods in which the
distribution of the primer composition of the totality of the
amplified cDNAs is analyzed with regard to groups 1)-4).
[0099] Examples of follow-up methods are:
[0100] A method for analyzing an RNA sample, preferably an mRNA
sample, which comprises
[0101] a) using a first primer, which is, where appropriate,
labeled with a first dye, to prepare the first strand of a
complementary DNA sample (cDNA sample) from an RNA sample,
preferably an mRNA sample,
[0102] b) using a second primer, which is preferably labeled with a
second dye, to prepare the second strand of this cDNA sample,
[0103] c) using the first primer, which is labeled with a first
dye, and the second primer, which is labeled with a second dye, to
amplify the cDNA sample, and
[0104] d) analyzing the composition of the amplified and labeled
cDNA sample, and
[0105] e) determining the primer composition of amplified
cDNAs.
[0106] A method for analyzing an RNA sample, preferably an mRNA
sample, which comprises
[0107] a) using a first primer, which is, where appropriate,
labeled with a first dye, to prepare the first strand of a
complementary DNA sample (cDNA sample) from an RNA sample,
preferably an mRNA sample,
[0108] b) using a second primer, which is preferably labeled with a
second dye, to prepare the second strand of this cDNA sample,
[0109] c) using the first primer, which is labeled with a first
dye, and the second primer, which is labeled with a second dye, to
amplify the cDNA sample, and
[0110] d) analyzing the composition of the amplified and labeled
cDNA sample,
[0111] e) determining the primer composition of amplified cDNAs,
and
[0112] f) selecting cDNAs having a particular primer composition,
preferably those cDNAs which contain both the first and the second
primer, and subjecting them to further analysis.
[0113] The RNA or mRNA samples which are used for the comparative
analysis or methods can, for example, be isolated from different
cells whose expression patterns are to be compared with each other.
In this context, the mRNA samples can, for example, be mRNA samples
which are derived from different cells or cell types which differ
from each other in their stage of differentiation and/or
development or in the stage of the cell cycle or which have a
different history (e.g. different culture conditions (e.g. pH,
temperature or composition), or which have been treated with
pharmacological active compounds or
disease-promoting/disease-inhibiting or disease-inducing substances
(e.g. carcinogenic or mutagenic substances) or have been exposed
thereto (e.g. UV light)). Such RNA or mRNA samples can be compared
with RNA or mRNA samples which have not been exposed, or not
exposed to this extent, to these substances. The RNA or mRNA
samples can be isolated from particular tissues. For example,
healthy tissue can be compared analytically with pathological
tissue, young tissue with old tissue, treated tissue with untreated
tissue, and induced tissue with non-induced tissue, while tissues
from different stages of development and/or the cell cycle and/or
differentiation can also be compared analytically, with tissue
denoting tissue, organ, cell type, culture-derived cells, cells of
a cell line or the cells of an individual.
[0114] The method can be used for analyzing differential gene
expression (in a tissue or in a cell). In particular, the method
can be used for analytically comparing differential gene expression
(in two or more tissues or cells).
[0115] The method can be used for identifying and/or characterizing
pharmacological active compounds. Furthermore, the method can be
used for identifying and/or characterizing target genes or target
proteins. These target genes or target proteins are, in particular,
genes or proteins which have a function (which is as specific as
possible) in the prevention, origin and/or progress and/or healing
of a disease, in the differentiation process, e.g. cell
differentiation (where appropriate, before or after induction)
and/or in the differentiation process of a disease, in the cell
cycle or cell cycle control and/or cell development, e.g. the
process of cell aging.
[0116] The method is seen to be advantageous in comparison with
conventional radioactive DDRT since the use of fluorescence-labeled
primers can be substituted for the use of radioactively labeled
nucleotides or radioactively labeled primers. In the novel method
(DDRT variant) which is described here, in contrast to the known
fluorescence DDRTs, each amplified cDNA is labeled by using two
primers which are differentially labeled, preferably with
fluorescences having different excitation spectra. In this way, by
using an appropriate evaluation method, it is possible to detect
each amplified, labeled cDNA, i.e. the complexity of the amplified
cDNA is retained and is detectable (products 1), 2), 3), and 4) can
be detected; e.g. the band complexity in the gel is retained). In
addition to this, it is possible to draw unambiguous conclusions
about the primer composition of an amplified labeled cDNA (i.e. it
is possible to assign the cDNA to 1), 2), 3) and 4)). This makes it
possible to select the cDNAs which are to be subjected to further
analysis because of their primer composition, e.g. to select
amplified cDNAs which actually do derive from the 3' regions of the
gene sequences. When this method, which is based on using two
differently labeled primers, is employed, it is possible,
therefore, to reduce the time and expense involved in possibly
having to isolate and analyze redundant cDNAs. Despite this, all
the amplified cDNAs are detected, and can be analyzed,
simultaneously, as in conventional radioactive DDRT, which is a
feature which is not afforded in conventional fluorescence
DDRT.
[0117] The invention also relates to a test kit for implementing
the method.
EXAMPLES
[0118] The enzymes employed are obtained from Gibco BRL/Life
Technologies (Karlsruhe, Germany) (reverse transcriptase and Taq
polymerase) and Promega (Heidelberg) (RNAsin).
[0119] Thermocycler: Perkin Elmer GeneAmp PCR System 2400 (Perkin
Elmer, Weiterstadt).
Example 1
[0120] Reverse Transcription
[0121] Reaction mixture: 1 .mu.l of RNA (100 ng-1 .mu.g of total
RNA or 1 ng-10 ng of poly-A-RNA/mRNA), 1 .mu.l of primer 1 (10
.mu.M primer 1, e.g. (T).sub.X-MA-3' or Cy5-(T).sub.XMA-3', where
M=A, C, G and X=11-15), and 8 .mu.l of H.sub.2O (nuclease-free).
The reaction mixture was incubated at 70.degree. C. for 5 min and
then placed on ice.
[0122] 4 .mu.l of 5-times RT buffer (Gibco BRL), 2 .mu.l of DTT
[0.1M], 1 .mu.l of SuperScript reverse transcriptase [200 U/.mu.l]
(Gibco BRL), 1 .mu.l of RNAsin [40 U/.mu.l] and 2 .mu.l of dNTP
[250 .mu.M] were then added.
[0123] The reverse transcription was carried out at
37.degree.-50.degree. C. for 60 min and the enzyme was then
inactivated at 70.degree. C. (10 min).
Example 2
[0124] PCR
[0125] 2 .mu.l of 10-times PCR buffer (Gibco BRL), 0.9 .mu.l of W-1
detergent [1%] (Gibco BRL), 0.75 .mu.l of MgCl.sub.2 [50 mM] (Gibco
BRL), 1.6 .mu.l of dNTP [250 .mu.M], 0.5 .mu.l of Taq DNA
polymerase [5 U/.mu.l] (Gibco BRL), 1 .mu.l each of primer 1 and
primer 2 ([10.mu.M]; e.g. Cy5-T7-(T).sub.X-MA-3' as primer 1 (where
"T7" is a sequence segment from the T7 promoter) and
fluorescein-(N).sub.X, where X=10-25 and N=A, C, G, T) and 10.25
.mu.l of H.sub.2O were added to 2 .mu.l of the RT mixture from
Example 1.
[0126] The PCR was carried out under the following conditions:
2 5 min at 94.degree. C. 40 .times.: 1 min at 94.degree. C., 2 min
at 40.degree. C.-60.degree. C., 1 min at 72.degree. C. 7 min at
72.degree. C.
[0127] After the conclusion of the PCR, the reaction mixture is
stored at 4.degree. C. and then fractionated on a sequencing gel
(5% polyacrylamide; 8 M urea).
[0128] The labeled amplified cDNAs can be detected, for example,
using a scanner which excites at a wavelength of 430+/-30 nm (for
fluorescein: excitation wavelength 490 nm, emission wavelength 520
nm) and 635+/-5 nm (for Cy5: excitation wavelength 650 nm, emission
wavelength 675 nm). The Molecular Dynamics Storm Imager is an
example of this type of scanner.
Sequence CWU 1
1
2 1 14 DNA Artificial Sequence exon (1)..(14) Description of
Artificial Sequence synthetic "V=A,C,G; N=A,C,G,T" 1 tttttttttt
ttvn 14 2 17 DNA Artificial Sequence exon (1)..(17) Description of
Artificial Sequence synthetic "V=A,C,G; N=A,C,G,T" 2 tttttttttt
tttttvn 17
* * * * *